Pre-mRNA splicing is an ubiquitous feature of eukaryotic gene expression. Introns must be properly removed for most protein synthesis to take place correctly. They are recognized by trans-acting factors during the early phases of spliceosome assembly, which commits the pre- mRNA substrate to the splicing pathway. Splice sites and the branchpoint sequence are identified at these early splicing steps, and their identification probably contributes to the fidelity of the splicing process as well as to the choice of splicing partners, namely, which exons are chosen to be ligated together. Many diseases are caused by splicing errors, and the regulation of viral gene expression during HIV infection is dependent upon aspects of this pre-mRNA recognition system. In both yeast and mammalian systems, U1 snRNP binding to pre-mRNA is an important step in these early events. Yet many aspects of these events, including the U1 snRNP-pre-mRNA interaction, remain poorly characterized. To better understand the early stages of splicing, we will use genetic, molecular genetic, and biochemical techniques in yeast (S. cerevisiae) to characterize U1 snRNP, associated and interacting factors, and the U1 snRNP-pre-mRNA complex that forms in a yeast whole cell splicing extract. Five proteins splicing factors are of most interest: U1C, Mud1p (the years U1A protein), CBC (the yeast cap binding complex with its two subunits CBP20 and CBP80), Mud2p (the yeast protein most similar to human U2AF65), and BBP (the branchpoint bridging factor, the yeast orthologue of the mammalian splicing factor SF1). BBP and Mud2p probably collaborate to effect branchpoint recognition, which will be studied. In vivo, we will visualize the association of pre-mRNA and U1 snRNP during transcription, i.e., on nascent RNA. We will also more generally study the structure of RNA within RNP, the role of HnRNP proteins and the other proteins associated with individual transcripts under different circumstances.